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Hairy cell leukemia variant (HCL-v) is a rare malignancy of clonal mature B-cells that follows a chronic disease course. HCL-v patients are often resistant to purine nucleoside analogs, which are the first-line therapy. To address the shortcomings of current therapy for HCL-v, we investigated the activity of a BAFF ligand-based CAR-T cell which binds to all three BAFF receptors, BAFF-receptor, TACI, and BCMA. Here, we demonstrate that HCLv patient-derived cells highly express all three BAFF receptors and that BAFF CAR-T cells induce significant cytotoxicity in vitro against both cell lines and HCL-v patient cells. This cytotoxicity corresponds with significant CAR-T cell activation, degranulation, and release of pro-inflammatory cytokines after co-incubation with HCLv cells. Furthermore, we successfully generated BAFF CAR-T cells directly from an HCLv patient and observed direct autologous killing against patient tumor cells in vitro. These HCLv patient-derived CAR-T cells were also effective in killing the Hair-M cell line and tumor cells derived from a different HCLv patient. Lastly, we also developed two mouse xenograft models for HCL, a subcutaneous Bonna-12 model and intravenous Hair-M xenograft model. We observed decreases in tumor burden and prolonged overall survival without significant toxicity. In conclusion, here we show that BAFF CAR-T cells exert anti-tumor effects in vitro and in vivo against multiple cell lines and patient-derived HCL-v samples and may be a successful therapeutic strategy for HCLv patients.

Despite success in treating some hematological malignancies, CAR-T cells have not yet produced similar outcomes in solid tumors due, in part, to the tumor microenvironment, poor persistence, and a paucity of suitable target antigens. Importantly, the impact of the CAR components on these challenges remains focused on the intracellular signaling and antigen-binding domains. In contrast, the flexible hinge and transmembrane domains have been commoditized and are the least studied components of the CAR. Here, we compared the hinge and transmembrane domains derived from either the CD8ɑ or CD28 molecule in identical GUCY2C-targeted third-generation designs for colorectal cancer. While these structural domains do not contribute to differences in antigen-independent contexts, such as CAR expression and differentiation and exhaustion phenotypes, the CD8ɑ structural domain CAR has a greater affinity for GUCY2C. This results in increased production of inflammatory cytokines and granzyme B, improved cytolytic effector function with low antigen-expressing tumor cells, and robust anti-tumor efficacy in vivo compared with the CD28 structural domain CAR. This suggests that CD8α structural domains should be considered in the design of all CARs for the generation of high-affinity CARs and optimally effective CAR-T cells in solid tumor immunotherapy.

Chimeric antigen receptor (CAR) T cells have great potential in cancer therapy, particularly in treating hematologic malignancies. However, their efficacy in solid tumors remains limited, with a significant proportion of patients failing to achieve long-term complete remission. One major challenge is the premature exhaustion of CAR-T cells, often due to insufficient metabolic energy. The survival, function and metabolic adaptation of CAR-T cells are key determinants of their therapeutic efficacy. We explore how targeting metabolic pathways in the tumor microenvironment can enhance CAR-T cell therapy by addressing metabolic competition and immunosuppression that impair CAR-T cell function. Tumors undergo metabolically reprogrammed to meet their rapid proliferation, thereby modulating metabolic pathways in immune cells to promote immunosuppression. The distinct metabolic requirements of tumors and T cells create a competitive environment, affecting the efficacy of CAR-T cell therapy. Recent research on glucose, lipid and amino acid metabolism, along with the interactions between tumor and immune cell metabolism, has revealed that targeting these metabolic processes can enhance antitumor immune responses. Combining metabolic interventions with existing antitumor therapies can fulfill the metabolic demands of immune cells, providing new ideas for tumor immunometabolic therapies. This review discusses the latest advances in the immunometabolic mechanisms underlying tumor immunosuppression, their implications for immunotherapy, and summarizes potential metabolic targets to improve the efficacy of CAR-T therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Microplastics (MPs) are increasingly recognized as contaminants present in various environments and are widely acknowledged as potential hazards to the mammalian immune system. In our study of chimeric antigen receptor T cell (CAR-T) therapy, we observed the presence of MP in CAR-T cell products for the first time. It is worth exploring whether MP could enter CAR-T cells and how they might affect CAR-T cells' functionality. Therefore, we analyzed how MP affected CD19 and BCMA-CAR-T cells. Based on flow cytometry, ELISA, and cytotoxicity analysis of in vitro and in vivo experiments, MP suppressed the activity of CAR-T cells. Subsequent investigation revealed that the exposure of CAR-T cells to varying concentrations of MP resulted in a notable increase in apoptosis, ferroptosis, and exhaustion levels. Furthermore, the hyperactivation of the mTOR signaling pathway in MP-treated CAR-T cells was verified. The partial restoration of CAR-T cell function in MP was achieved by inhibiting the mTOR pathway. MP present a threat to CAR-T cell function due to their role in inducing CAR-T cell apoptosis, ferroptosis, and T-cell exhaustion through the hyperactivation of mTOR signaling pathways., Competing Interests: Declaration of Competing Interest Our authors claim no known personal relation or competing financial interests affecting this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although major progress has been made in the use of chimeric antigen receptor (CAR)-T-cell therapy for hematological malignancies, this method is ineffective against solid tumors largely because of the limited infiltration, activation and proliferation of CAR-T cells. To overcome this issue, we engineered CAR-T cells with synthetic Notch (synNotch) receptors, which induce local tumor-specific secretion of extracellular matrix (ECM)-degrading enzymes at the tumor site. SynNotch CAR-T cells achieve precise ECM recognition and robustly kill targeted tumors, with synNotch-induced enzyme production enabling the degradation of components of the tumor ECM. In addition, this regulation strongly increased the infiltration of CAR-T cells and the clearance of solid tumors, resulting in tumor regression without toxicity in vivo. Notably, synNotch CAR-T cells also promoted the persistent activation of CAR-T cells in patient-derived tumor organoids. Thus, we constructed a synthetic T-cell system that increases the infiltration and antitumor function of CAR-T cells, providing a strategy for targeting ECM-rich solid tumors., Competing Interests: Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest., (© 2024. The Author(s), under exclusive licence to CSI and USTC.)

Introduction: Ovarian cancer is the most malignant gynecological tumor. Previous studies have demonstrated that chimeric antigen receptor (CAR)-engineered NK-92 cells targeting folate receptor α (αFR) (NK-92-αFR-CAR) can specifically kill αFR-positive ovarian cancer cells. However, the migration barrier restricts antitumor effects of CAR-engineered cells., Objectives: To elucidate the mechanism by which NK-92-αFR-CAR cells induce the secretion of chemokine CXCL10 during killing ovarian cancer cells. It is speculated that NK-92-αFR-CAR-CXCR3A can target αFR and have chemotaxis of CXCL10, and they may have stronger killing effect of ovarian cancer., Methods: Study the mechanism of CXCL10 expression strongly induced by TNF-α and IFN-γ combined stimulation in ovarian cancer cells. Construct the fourth generation of NK-92-αFR-CAR-CXCR3A cells, which were co-expressed CXCR3A and αFR-CAR. Evaluate the killing and migration effects of NK-92-αFR-CAR-CXCR3A in vitro and in vivo., Results: RNA sequencing (RNA-seq) first revealed that the expression level of the chemokine CXCL10 was most significantly increased in ovarian cancer cells co-cultured with NK-92-αFR-CAR. Secondly, cytokine stimulation experiments confirmed that IFN-γ and TNF-α secreted by NK-92-αFR-CAR synergistically induced high CXCL10 expression in ovarian cancer cells. Further signaling pathway experiments showed that IFN-γ and TNF-α enhanced the activation level of the IFN-γ-IFNGR-JAK1/2-STAT1-CXCL10 signaling axis. Cytotoxicity experiments showed that NK-92-αFR-CAR-CXCR3A cells could not only efficiently kill αFR-positive ovarian cancer cells in vitro but also secrete IFN-γ and TNF-α. Higher migration than that of NK-92-αFR-CAR was detected in NK-92-αFR-CAR-CXCR3A using transwell assay. NK-92-αFR-CAR-CXCR3A effectively killed tumor cells in different mouse xenograft models of ovarian cancer and increased infiltration into tumor tissue., Conclusion: This study confirmed that IFN-γ and TNF-α secreted by αFR-CAR-engineered NK cells can synergistically induce high expression of CXCL10 in ovarian cancer cells and constructed self-driving αFR-CAR-engineered NK cells that can break through migration barriers based on CXCL10, which may provide a new therapeutic weapon for ovarian cancer., Competing Interests: Declaration of competing interest The authors have declared no conflict of interest, (Copyright © 2024. Published by Elsevier Inc.)

The remarkable success of mRNA-based coronavirus 2019 (COVID-19) vaccines has propelled the advancement of nanomedicine, specifically in the realm of RNA technology and nanomaterial delivery systems. Notably, significant strides have been made in the development of RNA-based in vivo chimeric antigen receptor (CAR) therapy. In comparison to the conventional ex vivo CAR therapy, in vivo CAR therapy offers several benefits including simplified preparation, reduced costs, broad applicability and decreased potential for carcinogenic effects. This review summarises the RNA-based CAR constructs in in vivo CAR therapy, discusses the current applications of in vivo delivery vectors and outlines the immune cells edited with CAR molecules. We aim for the conveyed messages to contribute towards the advancement of in vivo CAR application., (© 2024 John Wiley & Sons Ltd.)

CAR T-cell products targeting lineage-specific cell-of-origin antigens, thereby eliminating both tumor and healthy counterpart cells, are currently clinically approved therapeutics in B- and plasma-cell malignancies. While they represent a major clinical improvement, they are still limited in terms of efficacy by e.g. single, sometimes low-expressed antigen targeting, and in terms of safety by e.g., lack of on-off activity. Successful cell-of-origin non-discriminative targeting of heterogeneous hematopoietic stem and progenitor cell malignancies, such as acute myeloid leukemia (AML), will require antigen-versatile targeting and off-switching of effectors in order to then allow rescue by hematopoietic stem cell transplantation (HSCT), preventing permanent myeloablation. To address this, we developed adaptor-CAR (AdFITC-CAR) T-cells targeting fluoresceinated AML antigen-binding diabody adaptors. This platform enables the use of adaptors matching the AML-antigen-expression profile and conditional activity modulation. Combining adaptors significantly improved lysis of AML cells in vitro. In therapeutic xenogeneic mouse models, AdFITC-CAR T-cells co-administered with single diabody adaptors were as efficient as direct CAR T-cells, and combinatorial use of adaptors further enhanced therapeutic efficacy against both, cell lines and primary AML. Collectively, this study provides proof-of-concept that AdFITC-CAR T-cells and combinations of adaptors can efficiently enhance immune-targeting of AML., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Chimeric antigen receptor (CAR)-T therapy has demonstrated sustained clinical remission in numerous hematologic malignancies and has expanded to encompass solid tumors and autoimmune diseases. While progress is being made in establishing optimal culture conditions for CAR-T cells, the identification of the most effective cytokine for promoting their persistence in vitro remains elusive., Methods: Here, we employed scRNA-seq (single-cell RNA sequencing) analysis to investigate the potential alterations in biological processes within CAR-T cells following exposure to cytokines (IL-2, IL-12, and IL-21) and antigens. Transcriptomic changes in diverse CAR-T groups were compared following various treatments, with a focus on epigenetic modifications, metabolic shifts, cellular senescence, and exhaustion., Results: Our study reveals that CAR-T cells treated with antigen, IL-2, and IL-12 exhibit signs of exhaustion and senescence, whereas those treated with IL-21 do not display these characteristics. The activities of glycolysis and epigenetic changes were significantly increased by treatments with antigens, IL-2, and IL-12, while IL-21 treatment maintained the oxidative phosphorylation (OXPHOS) of CAR-T cells., Conclusions: Our findings suggest that IL-21 may play a role in preventing senescence and could be utilized in combination with other strategies, such as IL-2 and IL-12, for CAR-T culture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chimeric antigen receptor (CAR) T-cell therapy is highly effective for treating blood cancers such as B-cell malignancies, however, its effectiveness as an approach to treat solid tumors remains to be further explored. Here, we focused on the development of CAR-T cell therapies targeting tropomyosin-related kinase receptor B (TRKB), a highly expressed protein that is significantly associated with tumor progression, malignancy, and drug resistance in multiple forms of aggressive solid tumors. To achieve this, we screened brain-derived neurotrophic factor (BDNF) and neurotrophin 4 (NTF4) ligand-based CAR-T cells for their efficiency in targeting the TRKB receptor in the context of solid tumors, particularly hepatocellular carcinoma and pancreatic cancer. We demonstrated that TRKB is overexpressed not only in hepatocellular carcinoma and pancreatic carcinoma cell lines but also in cancer stem-like cells (CSCs). Notably, BDNF-CAR T and NTF4-CAR T cells could not only effectively target and kill TRKB-expressing pan-cancer cell lines in a dose-dependent manner but also effectively kill CSCs. We also performed in vivo studies to show that NTF4-CAR T cells have a better potential to inhibit the tumor growth of hepatocellular carcinoma xenografts in mice, compared with BDNF-CAR T cells. Taken together, our findings suggest that CAR-T targeting TRKB may be a promising approach for developing novel therapies to treat solid cancers., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Pancreatic cancer is known for its poor prognosis and resistance to conventional therapies, largely due to the presence of cancer stem cells (CSCs) and aggressive angiogenesis. Effectively targeting these CSCs and associated angiogenic pathways is crucial for effective treatment. This study leverages single-cell multi-omics to explore a novel therapeutic approach involving Chimeric Antigen Receptor (CAR) macrophages engineered to target the c-Met protein on pancreatic CSCs., Methods: We employed single-cell RNA sequencing to analyze pancreatic cancer tissue, identifying c-Met as a key marker of CSCs. CAR macrophages were engineered using a lentiviral system to express a c-Met-specific receptor. The phagocytic efficiency of these CAR macrophages against pancreatic CSCs was assessed in vitro, along with their ability to inhibit angiogenesis. The in vivo efficacy of CAR macrophages was evaluated in a mouse model of pancreatic cancer., Results: CAR macrophages demonstrated high specificity for c-Met + CSCs, significantly enhancing phagocytosis and reducing the secretion of angiogenic factors such as VEGFA, FGF2, and ANGPT. In vivo, these macrophages significantly suppressed tumor growth and angiogenesis, prolonging survival in pancreatic cancer-bearing mice., Conclusion: CAR macrophages targeting c-Met represent a promising therapeutic strategy for pancreatic cancer, offering targeted elimination of CSCs and disruption of tumor angiogenesis. This study highlights the potential of single-cell multi-omics in guiding the development of precision immunotherapies., Competing Interests: Declarations. Ethical statement: All animal experiments reported in this study were approved by the Animal Ethics Committee of Jiaxing University Medical Center (Approval No. JUMC2024-087). Consent for publication: Not Applicable. Consent for publication: The authors declare that this manuscript is original, has not been published before, and is not currently being considered for publication elsewhere. All authors have approved the manuscript and agree with its submission to Molecular Medicine. Competing interests: The authors declare no competing interests. Conflict of interest: The author declares no conflict of interest., (© 2024. The Author(s).)

Regenerative medicine is a rapidly expanding field harnessing human pluripotent stem cell (hPSC)-derived cells and tissues to treat many diseases, including type 1 diabetes. However, graft immune protection remains a key challenge. Chimeric antigen receptor (CAR) technology confers new specificities to effector T cells and immunosuppressive regulatory T cells (Tregs). One challenge in CAR design is identifying target molecules unique to the cells of interest. Here, we employ combinatorial genetic engineering to confer CAR-Treg-mediated localized immune protection to stem cell-derived cells. We engineered hPSCs to express truncated epidermal growth factor receptor (EGFRt), a biologically inert and generalizable target for CAR-Treg homing and activation, and generated CAR-Tregs recognizing EGFRt. Strikingly, CAR-Tregs suppressed innate and adaptive immune responses in vitro and prevented EGFRt-hPSC-derived pancreatic beta-like cell (sBC [stem cell-derived beta cell]) graft immune destruction in vivo. Collectively, we provide proof of concept that hPSCs and Tregs can be co-engineered to protect hPSC-derived cells from immune rejection upon transplantation., Competing Interests: Declaration of interests A provisional patent on the strategy described in this article has been submitted with L.M.R.F. and H.A.R. as inventors. L.M.R.F. is an inventor in and has received royalties from patents on genetically modified human stem cells and T cells and consults for Guidepoint Global and McKesson. H.A.R. holds patents in the regenerative medicine space and served as a science advisory board (SAB) member of Sigilon Therapeutics and Prellis Biologics and consults or has consulted for Sigilon Therapeutics, Eli Lilly, Minutia, Guidepoint Global, Axon Advisors, and Tolerance Bio. H.A.R. is a scientific cofounder of Tolerance Bio., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Targeting tumor-specific neoantigens holds promise for cancer immunotherapy, but their ultra-low expression on tumor cells poses challenges for T cell therapies. Here, we found that chimeric antigen receptors (CARs) exhibit 10-100 times lower sensitivity than T cell receptors (TCRs) when targeting human leukocyte antigen (HLA) class I-presented p53 R175H neoantigen. To enhance CAR functionality, we introduced T cell receptor fusion constructs (TRuCs) and synthetic TCRs and antigen receptors (STARs). Our data show that STARs optimally reproduce TCR antigen sensitivity, outperforming CARs and TRuCs in redirecting CD8 + and CD4 + T cells to recognize HLA class I neoantigens. STAR-T cells demonstrate superior killing of cancer cell lines with low neoantigen density in vitro and improved tumor control in mouse models compared to CAR-T and TRuC-T cells. These findings highlight CAR sensitivity limitations and present STARs as more effective synthetic receptors for T cell-based immunotherapy against tumors with low neoantigen density., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The application of chimeric antigen receptor (CAR) T cell therapy in solid tumors is hindered by life-threatening toxicities resulting from on-target, off-tumor killing of nonmalignant cells that express low levels of the target antigen. Mesothelin (MSLN) has been identified as a target antigen for CAR T cell treatment of mesothelioma, lung, ovarian, and other cancers because of its high expression on tumor cells and limited expression on mesothelial cells. However, fatal off-tumor toxicity of high-affinity MSLN-targeting CAR T cells has been reported in multiple clinical trials. In this study, we constructed CARs using mutant variants of a single-domain nanobody that bind both human and mouse MSLN with a wide range of affinities and examined tumor responses and their toxicities from on-target, off-tumor interactions in mouse models. CAR T cells with low nanomolar affinity (equilibrium dissociation constant, KD) exhibited profound systemic expansion with no apparent infiltration into the tumor. With a gradual reduction of CAR affinity toward the micromolar KD, the expansion of CAR T cells became more restricted to tumors. Our preclinical studies demonstrated that high-affinity MSLN CARs were associated with fatal on-target, off-tumor toxicity and that affinity-tuned CARs rendered T cells more selective for MSLN-high tumors.

Background: Oncofetal splice variants of extracellular matrix (ECM) proteins present a unique group of target antigens for the immunotherapy of pediatric cancers. However, limited data is available if these splice variants can be targeted with T cells expressing chimeric antigen receptors (CARs)., Methods: To determine the expression of the oncofetal version of tenascin C (TNC) encoding the C domain (C.TNC) in pediatric brain and solid tumors, we used quantitative reverse transcription PCR and immunohistochemistry. Genetically modified T cells were generated from human peripheral blood mononuclear cells and evaluated in vitro and in vivo., Results: We demonstrate that C.TNC is expressed on a protein level in pediatric tumors, including diffuse intrinsic pontine glioma, osteosarcoma, rhabdomyosarcoma, and Ewing sarcoma. We generate C.TNC-CAR T cells and establish that these recognize and kill C.TNC-positive tumor cells. However, their antitumor activity in vivo is limited. To improve the effector function of C.TNC-CAR T cells, we design a leucine zipper-based chimeric cytokine receptor that activates interleukin-18 signaling pathways (Zip18R). Expression of Zip18R in C.TNC-CAR T cells improves their ability to secrete cytokines and expand in repeat stimulation assays. C.TNC-CAR.Zip18R T cells also have significantly greater antitumor activity in vivo compared with unmodified C.TNC-CAR T cells., Conclusions: Our study identifies the C domain of the ECM protein TNC as a promising CAR T-cell therapy for pediatric solid tumors and brain tumors. While we focus here on pediatric cancer, our work has relevance to a broad range of adult cancers that express C.TNC., Competing Interests: Competing interests: EW, SL, JW, TIS, GK, JZ, and SG have patent applications in the fields of cell or gene therapy for cancer. MB, GK, and SG are coinventors on a patent application for the developed Zip receptor technology. SG is a member of the Scientific Advisory Board of Be Biopharma and CARGO, and the Data and Safety Monitoring Board (DSMB) of Immatics and has received honoraria from TESSA Therapeutics within the last year. The other authors declare no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The development of chimeric antigen receptor (CAR)-T cell therapies for solid tumors has attracted considerable attention, yet their clinical efficacy remains limited. Therefore, various efforts have been made to improve the efficacy of CAR-T cell therapy. As one promising strategy, incorporating the T-cell receptor (TCR) machinery into CAR structures has been reported to improve the efficacy of CAR-T cells in studies using conventional CARs targeting such as EGFR. However, in the case of peptide/major histocompatibility complex (pMHC)-targeted CARs, the advantages of exploiting TCR machinery have not been fully elucidated. We recently developed MAGE-A4-derived pMHC (MAGE-A4 pMHC)-targeted CAR-T cells (MA-CAR-T cells) using a highly specific human scFv antibody against MAGE-A4 p230-239 /HLA-A*02:01. We aimed to determine whether MAGE-A4 pMHC-targeted CAR-T cells using the TCR machinery (Hybrid MA-TCR-T cells) exhibit superior functionality without compromising antigen specificity., Methods: We constructed a retroviral vector expressing Hybrid MA-TCR where MAGE-A4 pMHC-specific scFv are fused to human TCR constant chains., Results: Hybrid MA-TCR-T cells demonstrated superior in vitro functions compared with MA-CAR-T cells, while maintaining strict antigen specificity. In addition, functional superiority of Hybrid MA-TCR-T cells to MA-CAR-T cells became more pronounced on repetitive antigen stimulation. In particular, Hybrid MA-TCR-T cells significantly inhibited tumor growth in an immunodeficient mouse model more effectively than MA-CAR-T cells. Ex vivo analyses indicated that their enhanced therapeutic efficacy might result from higher infiltration of functionally active, less differentiated Hybrid MA-TCR-T cells in tumor tissues., Conclusions: These findings suggest that leveraging the TCR machinery is a promising strategy for enhancing pMHC-targeted CAR-T cell therapy for solid tumors, potentially leading to more effective treatments., Competing Interests: Competing interests: The Department of Personalized Cancer Immunotherapy, Mie University Graduate School of Medicine, is an endowment department supported by a grant from T Cell Nouveau Inc. NI and KN are employees of T Cell Nouveau Inc., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

NKG2D ligands (NKG2DLs) are broadly expressed in cancer. To target these, we describe an adaptor chimeric antigen receptor (CAR) termed NKG2D/Dap10-12. Herein, T cells are engineered to co-express NKG2D with a fusion protein that comprises Dap10 joined to a Dap12 endodomain. NKG2D/Dap10-12 T cells elicit compelling efficacy, eradicating or controlling NKG2DL-expressing tumors in several established xenograft models. Importantly, durable responses, long-term survival, and rejection of tumor re-challenge are reproducibly achieved. Efficacy is markedly superior to a clinical stage CAR analog, comprising an NKG2D-CD3ζ fusion. Structure-function analysis using an extended CAR panel demonstrates that potency is dependent on membrane proximity of signaling units, high NKG2D cell surface expression, adaptor structure, provision of exogenous Dap10, and inclusion of one rather than three immune tyrosine activation motifs per signaling unit. Potent therapeutic impact of NKG2D/Dap10-12 T cells is also underpinned by enhanced oxidative phosphorylation, reduced senescence, and transcriptomic re-programming for increased ribosomal biogenesis., Competing Interests: Declaration of interests J.M. is CSO, scientific founder, and shareholder of Leucid Bio. M.G., F.K., C.M.H., R.M., C.B., C.T., and D.M.D. are employees of Leucid Bio while P.D. is a former Leucid Bio employee. D.L.-Y. is undertaking a PhD studentship funded by Leucid Bio and has acted as a consultant for Leucid Bio. J.M., D.M.D., D.L.-Y., and F.K. are co-inventors on patent filings in relation to adaptor CAR technology., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Glioblastoma is the most common lethal primary brain tumor, urging evaluation of new treatment options. Chimeric antigen receptor (CAR)-T cells targeting B7 homolog 3 (B7-H3) are promising because of the overexpression of B7-H3 on glioblastoma cells but not on healthy brain tissue. Nanobody-based (nano)CARs are gaining increasing attention as promising alternatives to classical single-chain variable fragment-based (scFv)CARs, because of their single-domain nature and low immunogenicity. Still, B7-H3 nanoCAR-T cells have not been extensively studied in glioblastoma., Methods: B7-H3 nanoCAR- and scFvCAR-T cells were developed and evaluated in human glioblastoma models. NanoCAR-T cells targeting an irrelevant antigen served as control. T cell activation, cytokine secretion and killing capacity were evaluated in vitro using ELISA, live cell imaging and flow cytometry. Antigen-specific killing was assessed by generating B7-H3 knock-out cells using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-genome editing. The tumor tracing capacity of the B7-H3 nanobody was first evaluated in vivo using nuclear imaging. Then, the therapeutic potential of the nanoCAR-T cells was evaluated in a xenograft glioblastoma model., Results: We showed that B7-H3 nanoCAR-T cells were most efficient in lysing B7-H3 pos glioblastoma cells in vitro. Lack of glioblastoma killing by control nanoCAR-T cells and lack of B7-H3 neg glioblastoma killing by B7-H3 nanoCAR-T cells showed antigen-specificity. We showed in vivo tumor targeting capacity of the B7-H3 nanobody-used for the nanoCAR design-in nuclear imaging experiments. Evaluation of the nanoCAR-T cells in vivo showed tumor control in mice treated with B7-H3 nanoCAR-T cells in contrast to progressive disease in mice treated with control nanoCAR-T cells. However, we observed limiting toxicity in mice treated with B7-H3 nanoCAR-T cells and showed that the B7-H3 nanoCAR-T cells are activated even by low levels of mouse B7-H3 expression., Conclusions: B7-H3 nanoCAR-T cells showed promise for glioblastoma therapy following in vitro characterization, but limiting in vivo toxicity was observed. Off-tumor recognition of healthy mouse tissue by the cross-reactive B7-H3 nanoCAR-T cells was identified as a potential cause for this toxicity, warranting caution when using highly sensitive nanoCAR-T cells, recognizing the low-level expression of B7-H3 on healthy tissue., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chimeric antigen receptor T (CAR-T) cells show remarkable efficacy for some hematological malignancies. However, CAR targets that are expressed at high level and selective to tumors are scarce. Several strategies have been proposed to tackle the on-target off-tumor toxicity of CAR-T cells that arise from suboptimal selectivity, but these are complicated, with many involving dual gene expression for specificity. In this study, we show that switchable CAR-T cells with a tumor targeting adaptor can mitigate on-target off-tumor toxicity against a low selectivity tumor antigen that cannot be targeted by conventional CAR-T cells, such as CD40. Our system is composed of anti-cotinine murine CAR-T cells and cotinine-labeled anti-CD40 single chain variable fragments (scFv), with which we show selective tumor killing while sparing CD40-expressing normal cells including macrophages in a mouse model of lymphoma. Simple replacement of the tumor-targeting adaptor with a suicidal drug-conjugated tag may further enhance safety by enabling permanent in vivo depletion of the switchable CAR-T cells when necessary. In summary, our switchable CAR system can control CAR-T cell toxicity while maintaining therapeutic efficacy, thereby expanding the range of CAR targets., Competing Interests: Competing interests K.C. and E.Y.C. are founders and shareholders of Ticaros Inc. H.B.P., J.E.L., and G.R.J. are currently employees of Ticaros. K.C., J.C., H.B.P., K.H.K., S.I.K., and G.R.J. are co-inventors on the pending patent for anti-CD40 switchable CAR T cells. The other authors declare no competing interests., (© 2024. The Author(s).)

The redirection of T lymphocytes against tumor-associated or tumor-specific antigens, using bispecific antibodies or chimeric antigen receptors (CAR), has shown therapeutic success against certain hematological malignancies. However, this strategy has not been effective against solid tumors. Here, we describe the development of CAR T cells targeting p95HER2, a tumor-specific antigen found in HER2-amplified solid tumors. These CAR T cells display robust activity against p95HER2-expressing cell lines but demonstrate limited efficacy against patient-derived xenografts. As p95HER2 is invariably detectable on tumor cells that overexpress HER2, but not those that express HER2 at normal levels, we arm p95HER2-specific CAR T cells with affinity-tuned bispecific antibodies against HER2 and CD3 in order to redirect them only to HER2-amplified cells. The combination of p95HER2.CAR T cells and HER2 x CD3 bispecific antibodies lead to a complete regression in three HER2-positive, patient-derived mouse xenografts tumor models. This combination represents a promising strategy to redirect T cells against a subset of HER2-positive tumors., Competing Interests: Competing interests J.A. has received research funds from Roche, Byondis, Menarini and Molecular Partners and consultancy honoraria from Menarini, Mnemo and ARKIN. J.A. is an inventor of patent applications EP22382294, EP20382457, EP16191933.7, EP0930183.5 and P200801652. M.R.A., I.R.R., C.K., E.M., are inventors of patent application EP20382457. M.R.A., S.D., A.G.E., I.R.R., V.N. are inventors of patent application EP22382294. C.K. declares employment, patents and stock ownership with Roche. E.M. declares employment with Roche. E.G. reports: research agreements with Novartis, Roche, Thermo Fisher, AstraZeneca, Taiho, BeiGene, Janssen; consultant/advisor at Roche, Ellipses Pharma, Boehringer Ingelheim, Janssen Global Services, Seattle Genetics, Thermo Fisher, MabDiscovery, Anaveon, F-Star Therapeutics, Hengrui, Sanofi, Incyte; and payment or honoraria for speakers’ bureaus from Merck Sharp & Dohme, Roche, Thermo Fisher, Lilly, Novartis, SeaGen. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Background: Chimeric antigen receptor T (CAR-T) cell therapy has been shown remarkable efficacy in the treatment of hematological malignancies in recent years. However, a considerable proportion of patients would experience tumor recurrence and deterioration. Insufficient CAR-T cell persistence is the major reason for relapse. Multiple strategies to enhance the long-term antitumor effects of CAR-T cells have been explored and developed. In this study, we focused on tyrosine kinase inhibitors (TKIs), which have emerged immunomodulatory potential besides direct tumoricidal effects., Methods: Here, we screened 50 approved TKIs drugs and identified that afatinib (AFA) markedly enhanced the expressing of CD62L and inhibited reactive oxygen species level in T cells. And the underlying mechanisms of AFA medicating T cells were explored by detecting signal transduction, and metabolism pattern. Furthermore, we co-cultured AFA with CAR-T cells during the preparation stage and multianalyses of differentiation characteristics, metabolic profiling, and RNA sequencing revealed that AFA induce comprehensive metabolism remodeling and fate reprogramming. Based on it, we finally identified the antitumor efficacy of AFA-pretreatment CAR-T compared with negative-control CAR-T., Results: We identified that AFA blocked the T-cell receptor (TCR) and phosphoinositide 3-kinase-protein kinase B-mechanistic target of rapamycin signaling pathways, induced metabolic reprogramming and modulated T-cell differentiation. When combined with CAR-T cells, AFA inhibited the exhaustion and enhanced the persistence and cytotoxicity. Our results revealed that the pretreatment of AFA enables to boost CAR-T cells with strong antitumor cytotoxicity in leukemia mouse model., Conclusions: Our study systematically demonstrated that AFA pretreatment effectively enhanced CAR-T cells antitumor performance, which presents a novel optimization strategy for potent and durable CAR-T cell therapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

CAR-T therapy has revolutionized the field of oncology, offering a promising treatment option for cancer patients. However, the significant morbidity associated with therapy-related toxicity presents a major challenge to its widespread use. Despite extensive research into the underlying mechanisms of CAR-T therapy-related toxicity, there are still many unknowns. Furthermore, the lack of adequate in vitro models for assessing immunotoxicity and neurotoxicity further complicates the development of safer cellular therapies. Previously in our laboratory, we developed cancer-stroma spheres (CSS) composed of prostate adenocarcinoma PC3 cells and mesenchymal stem cells (MSC). Herein we present evidence that multicellular CSS could serve as a valuable in vitro model for toxicity studies related to CAR-T therapy. CSS containing CD19-overexpressing PC3M cells exhibited increased secretion of CAR-T cell toxicity-associated IL-8, MCP-1, and IP-10 in the presence of anti-CD19 CAR-T cells, compared to spheres derived from single cell types.

Background: Despite remarkable achievements in applying chimeric antigen receptor (CAR)-T cells to treat hematological malignancies, they remain much less effective against solid tumors, facing several challenges affecting their clinical use. We previously showed that multichain DNAX-activating protein (DAP) CAR structures could enhance the safety and efficacy of CAR-T cells when used against solid tumors. In particular, mesothelin (MSLN)-targeted CAR-T cell therapy has therapeutic potential in MSLN-positive solid tumors, including ovarian cancer and mesothelioma., Methods: In vitro cell killing assays and xenograft model were utilized to determine the anti-tumor efficacy of MSLN targeting DAP-CAR-T cells and other CAR-T cells. ELISA and flow cytometry analysis were used to assess the cytokine secretion capacity and proliferation ability. Eight patients with MSLN expression were enrolled to evaluate the safety and efficacy of MSLN-DAP CAR-T cell therapy. Single-cell sequencing was performed to explore the dynamics of immune cells in patients during treatment and to identify the transcriptomic signatures associated with efficacy and toxicity., Results: We found that multichain DAP-CAR formed by combining a natural killer cell immunoglobulin-like receptor truncator and DAP12 exhibited better cytotoxicity and tumor-killing capacity than other natural killer cell-activated receptors associated with DAP12, DAP10, or CD3Z. The safety and efficacy of MSLN-DAP CAR-T cell therapy in patients with ovarian cancer and mesothelioma were evaluated in a single-arm, open-label clinical trial (ChiCTR2100046544); two patients achieved partial response, while four patients had a stable disease status. Furthermore, single-cell sequencing analysis indicated that KT032 CAR-T cell infusion could recruit more immune cells and temporarily remodel the TME., Conclusions: Our study highlights the safety and therapeutic efficacy of multiple-chain DAP-CAR-T cell therapy targeting MSLN to treat patients with ovarian cancer and mesothelioma., Trial Registration: ChiCTR.org.cn, ChiCTR2100046544 . May 21, 2021., Competing Interests: Declarations Ethics approval and consent to participate This study was approved by the Institution Review Board of The First Affiliated Hospital of Nanjing Medical University (2021-SR-084). This study conformed to the principles of the Helsinki Declaration. Healthy donors or patients consented to participate in the study. All participants provided written informed consent prior to participation. The animal experiment in this study was approved by the Institutional Ethics Committee of Nanjing Medical University (IACUC2103036). Consent for publication All participants provided written informed consent for publication. Competing interests Authors EX-W, C-W, and JZ-Z were employed by Nanjing CART Medical Technology Co., Ltd. The other authors declare that they have no competing interests., (© 2024. The Author(s).)

CD8 + T cell exhaustion hampers control of cancer and chronic infections and limits chimeric antigen receptor (CAR) T cell efficacy. Targeting TET2 in CAR T cells provides therapeutic benefit; however, TET2's role in exhausted T cell (T EX ) development is unclear. In chronic lymphocytic choriomeningitis virus (LCMV) infection, TET2 drove conversion from stem cell-like T EX progenitors toward terminally differentiated and effector (T EFF )-like T EX . TET2 also enforced a terminally differentiated state in the early bifurcation between T EFF and T EX , indicating broad roles for TET2 in acquisition of effector biology. To exploit the therapeutic potential of TET2, we developed clinically actionable TET2- targeted CAR T cells by disrupting TET2 via knock-in of a safety switch alongside CAR knock-in at the TRAC locus. TET2 -targeted CAR T cells exhibited restrained terminal exhaustion in vitro and enhanced antitumor responses in vivo. Thus, TET2 regulates fate transitions in T EX differentiation and can be targeted with a safety mechanism in CAR T cells for improved tumor control.

Purpose: Greater disease burden is a well-established predictor of poorer outcomes following chimeric antigen receptor T-cell (CAR T) therapy. Although bridging therapy (BT) is widely used between leukapheresis and CAR T infusion, limited data have evaluated the impact of BT on CAR T outcomes. In this study, we hypothesized that the quantitative dynamics of radiomic cytoreduction during bridging are prognostic., Experimental Design: Patients with large B-cell lymphoma treated with CD19-CAR T from 2016 to 2022 were included in the study. Metabolic tumor volume (MTV) was determined for all patients on pre-leukapheresis PET and on post-BT/pre-infusion PET in those who received BT. Patients were stratified into "High" and "Low" disease burden using an MTV cutpoint of 65.4cc established by maximally selected log-rank statistic for progression-free survival (PFS)., Results: Of 191 patients treated with CAR T, 144 (75%) received BT. In the BT cohort, 56% had a reduction in MTV post-BT. On multivariate analysis, the MTV trajectory across the bridging period remained significantly associated with PFS (P < 0.001); however, notably, patients with improved MTV (High->Low) had equivalent PFS compared with those with initially and persistently low MTV (Low->Low; HR for High->Low MTV: 2.74; 95% confidence interval, 0.82-9.18). There was a reduction in any grade immune effector cell-associated neurotoxicity syndrome in the High->Low MTV cohort as compared with the High->High MTV cohort (13% vs. 41%; P = 0.05)., Conclusions: This is the first study to use radiomics to quantify disease burden pre- and post-BT in a large real-world large B-cell lymphoma cohort. We demonstrate that effective BT can enable initially high-disease burden patients to achieve post-CAR T outcomes comparable with low-disease burden patients., (©2024 American Association for Cancer Research.)

CAR T cell therapy has been an effective treatment option for hematological malignancies. However, the therapeutic potential of CAR T cells can be reduced by several constraints, partly due to immunogenicity and toxicities. The lack of established workflows enabling thorough evaluation of new candidates, limits comprehensive CAR assessment. To improve the selection of lead CAR candidates, we established a stringent, multistep workflow based on specificity assessments, employing multiple assays and technologies. Moreover, we characterized a human FOLR1-directed CAR binding domain. Selection of binding domains was based on extensive specificity assessment by flow cytometry and imaging, to determine on-/off-target and off-tumor reactivity. CAR T cell functionality and specificity were assessed by high-throughput screening and advanced in vitro assays. Our validation strategy highlights that assays comprehensively characterizing CAR functionality and binding specificity complement each other. Thereby, critical specificity considerations can be addressed early in the development process to overcome current limitations for future CAR T cell therapies.

Background: Neoadjuvant therapy with trastuzumab, pertuzumab and paclitaxel (THP) has significantly improved the prognosis of patients with human epidermal growth factor receptor 2 (HER2) + breast cancer (BC). However, there remains a subset of non-responsive patients. Thus, this study sought to identify key regulators of THP neoadjuvant therapy resistance and potential targets to sensitize sensitivity., Methods: The Cancer Genome Atlas database, Gene Expression Omnibus and membrane protein database were used to identify the key regulator of THP neoadjuvant resistance. The biological functions and mechanisms of delta-like 4 proteins (DLL4) in THP therapy resistance were investigated in vitro and in vivo using the bioinformatic analysis, multiplex immunofluorescence, flow cytometry, sphere formation assays and chromatin immunoprecipitation, etc. Furthermore, DLL4-targeted chimeric antigen receptor (CAR)-T cells were established to sensitize THP therapy., Results: DLL4 was identified as a key target in THP neoadjuvant therapy resistance for HER2 + BC. Mechanistically, DLL4 + tumor cells exhibited enhanced stemness and resistance to the THP neoadjuvant chemotherapy. Additionally, soluble DLL4 can split away from tumor cells and diffuse into the stroma, where it can activate the Notch signaling pathway in neutrophils, inducing the formation and release of neutrophil extracellular traps (NETs) by regulating the transcription of MPO, PDIA4 and ELANE. This led to the exclusion of lymphocyte infiltration, thereby enhancing therapy resistance. What is more, we designed a DLL4-targeted CAR-T to eliminate DLL4 + tumor cells and reverse the resistant status., Conclusions: Our study revealed novel functions of DLL4 in cell stemness and immune infiltration, including NET formation and T cell exclusion, which collectively contributed to THP neoadjuvant therapy resistance in HER2 + BC. Furthermore, we provided a CAR-T-based therapy to sensitize the THP neoadjuvant therapy., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Recent times have witnessed remarkable progress in cancer immunotherapy, drastically changing the cancer treatment landscape. Among the various immunotherapeutic approaches, adoptive cell therapy (ACT), particularly chimeric antigen receptor (CAR) T cell therapy, has emerged as a promising strategy to tackle cancer. CAR-T cells are genetically engineered T cells with synthetic receptors capable of recognising and targeting tumour-specific or tumour-associated antigens. By leveraging the intrinsic cytotoxicity of T cells and enhancing their tumour-targeting specificity, CAR-T cell therapy holds immense potential in achieving long-term remission for cancer patients. However, challenges such as antigen escape and cytokine release syndrome underscore the need for the continued optimisation and refinement of CAR-T cell therapy. Here, we report on the challenges of CAR-T cell therapies and on the efforts focused on innovative CAR design, on diverse therapeutic strategies, and on future directions for this emerging and fast-growing field. The review highlights the significant advances and changes in CAR-T cell therapy, focusing on the design and function of CAR constructs, systematically categorising the different CARs based on their structures and concepts to guide researchers interested in ACT through an ever-changing and complex scenario. UNIVERSAL CARs, engineered to recognise multiple tumour antigens simultaneously, DUAL CARs, and SUPRA CARs are some of the most advanced instances. Non-molecular variant categories including CARs capable of secreting enzymes, such as catalase to reduce oxidative stress in situ, and heparanase to promote infiltration by degrading the extracellular matrix, are also explained. Additionally, we report on CARs influenced or activated by external stimuli like light, heat, oxygen, or nanomaterials. Those strategies and improved CAR constructs in combination with further genetic engineering through CRISPR/Cas9- and TALEN-based approaches for genome editing will pave the way for successful clinical applications that today are just starting to scratch the surface. The frontier lies in bringing those approaches into clinical assessment, aiming for more regulated, safer, and effective CAR-T therapies for cancer patients.

Background: Immunotherapy, such as chimeric antigen receptor T (CAR-T) cells targeting CD33 or CD123, has been well developed over the past decade for the treatment of acute myeloid leukemia (AML). However, the inability to sustain tumor-free survival and the possibility of relapse due to antigen loss have raised concerns. A dual targeting of CD33 and CD123 is needed for better outcomes., Methods: Based on our previously constructed CD33 and CD123 monovalent CAR-T, Loop33 × 123 and Loop123 × 33 CAR-T were constructed with molecular cloning techniques. All CAR-T cells were generated by lentivirus transduction of T cells from healthy donors. Phenotype detection was evaluated on day 7 concerning activation, exhaustion, and subtype proportions. Coculture killing assays were conducted using various AML cell lines and primary AML cells. Degranulation and cytokine secretion levels were detected by flow cytometry. Cell-derived xenograft models were established using wild-type Molm 13 cell lines, or a mixture of Molm 13-KO33 and Molm 13-KO123 cells as an ideal model of immune escape. By monitoring body weight and survival of tumor-bearing mice, Loop33 × 123 and Loop123 × 33 CAR-T cells were further assessed for their efficacy in vivo., Results: In vitro study, our results demonstrated that Loop33 × 123 CAR-T cells could efficiently eliminate AML cell lines and primary AML cells with elevated degranulation and cytokine secretion levels. Compared with our previously constructed monovalent CD33 or CD123 CAR-T cells, Loop33 × 123 CAR-T cells showed superior advantages in an immune escape model. In vivo studies further confirmed that Loop33 × 123 CAR-T cells could effectively prolong the survival of mice without significant toxicity. However, Loop123 × 33 CAR-T cells failed to show the same effects. Furthermore, Loop33 × 123 CAR-T cells efficiently circumvented potential immune escape, a challenge where monovalent CAR-T cells failed., Conclusions: Loop33 × 123 CAR-T targeting CD33 and CD123 could efficiently eliminate AML cells and prolong survival of tumor-bearing mice, while addressing the issue of immune escape., Competing Interests: Declarations Conflict of interest The authors declare no competing interests., (© 2024. The Author(s).)

To address immune escape, multi-specific CAR-T-cell strategies use natural ligands that specifically bind multiple receptors on malignant cells. In this context, we propose a split CAR design comprising a universal receptor expressed on T cells and ligand-based switch molecules, which preserves the natural trimeric structure of ligands like APRIL and BAFF. Following optimization of the hinges and switch labeling sites, the split-design CAR-T cells ensure the native conformation of ligands, facilitating the optimal formation of immune synapses between target cancer cells and CAR-T cells. Our CAR-T-cell strategy demonstrates antitumor activities against various B-cell malignancy models in female mice, potentially preventing immune escape following conventional CAR-T-cell therapies in the case of antigen loss or switching. This ligand-based split CAR design introduces an idea for optimizing CAR recognition, enhancing efficacy and potentially improving safety in clinical translation, and may be broadly applicable to cellular therapies based on natural receptors or ligands., Competing Interests: Competing interests The Chinese patent “A method to prepare natural ligand-mediated switchable gene-modified immune cells against multiple targets” with the application number CN202010073487.1 is related to the present work. The applicant for the patent is Peking University Shenzhen Graduate School. The authors declare that they have no competing interests., (© 2024. The Author(s).)

Background: The failure of conventional therapies and the propensity for recurrence and metastasis make triple-negative breast cancer (TNBC) a formidable challenge with grim prognoses and diminished survival rates. Immunotherapy, including immune checkpoint blockade and chimeric antigen receptor (CAR)-T cell therapy, presents innovative and potentially more effective strategies for addressing TNBC. Within this context, the inducible costimulator (ICOS), a member of the CTLA4/CD28 family, plays a crucial role in regulating immune responses and T-cell differentiation by binding to its ligand ICOSL. However, the impact of the ICOS/ICOSL axis on cancer varies., Methods: In this study, immunohistochemistry was conducted to examine the expression level of ICOSL in TNBC tumor tissues. We developed ICOS-enhanced B7H3-CAR-T cells (ICOS-B7H3-CAR) using the third-generation CAR-T cell technology, which featured magnified ICOS expression and targeted the B7H3 antigen. Xenograft and metastasis models of TNBC were conducted to examine the cytotoxicity and durability of CAR-T cells in tumors. Overexpression and CRISPR/Cas9-mediated knockout (KO) techniques were employed to regulate the expression of ICOSL on TNBC cell lines., Results: Notably, we observed elevated ICOSL expression in TNBC tumor tissues, which correlated with poor survival prognosis in patients with TNBC. Compared with conventional B7H3-CAR-T cells, ICOS-B7H3-CAR-T cells significantly inhibited the tumor growth of TNBC cells both in vitro and in vivo, accompanied by increased secretion of cytokines such as interferon gamma and tumor necrosis factor alpha. Furthermore, the in vivo experiments illustrated that ICOS-B7H3-CAR-T cells exhibited prolonged antitumor activity and could effectively eradicate metastases in a TNBC metastasis model, consequently extending survival. Importantly, manipulating the expression of ICOSL on TNBC cells through overexpression or KO significantly influenced the function of ICOS-B7H3-CAR-T cells. This suggests that the level of ICOSL expression on TNBC cells is critical for enhancing the potent antitumor effects of ICOS-B7H3-CAR-T cells., Conclusion: Overall, our study highlights the potential clinical application of ICOS as a promising strategy for combating TNBC recurrence and metastasis., Competing Interests: Competing interests: No, there are no competing interests., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

A significant challenge for chimeric antigen receptor (CAR) T cell therapy against glioblastoma (GBM) is its immunosuppressive microenvironment, which is densely populated by protumoral glioma-associated microglia and macrophages (GAMs). Myeloid immune checkpoint therapy targeting the CD47-signal regulatory protein alpha (SIRPα) axis induces GAM phagocytic function, but CD47 blockade monotherapy is associated with toxicity and low bioavailability in solid tumors. In this work, we engineer a CAR T cell against epidermal growth factor receptor variant III (EGFRvIII), constitutively secreting a signal regulatory protein gamma-related protein (SGRP) with high affinity to CD47. Anti-EGFRvIII-SGRP CAR T cells eradicate orthotopic EGFRvIII-mosaic GBM in vivo, promoting GAM-mediated tumor cell phagocytosis. In a subcutaneous CD19 + lymphoma mouse model, anti-CD19-SGRP CAR T cell therapy is superior to conventional anti-CD19 CAR T. Thus, combination of CAR and SGRP eliminates bystander tumor cells in a manner that could overcome main mechanisms of CAR T cell therapy resistance, including immune suppression and antigen escape., (© 2024. The Author(s).)

Acute myeloid leukemia (AML) is a malignant cancer characterized by abnormal differentiation of hematopoietic stem and progenitor cells (HSPCs). While chimeric antigen receptor T (CAR-T) cell immunotherapies target AML cells, they often induce severe on-target/off-tumor toxicity by attacking normal cells expressing the same antigen. Here, we used base editors (BEs) and a prime editor (PE) to modify the epitope of CD123 on HSPCs, protecting healthy cells from CAR-T-induced cytotoxicity while maintaining their normal function. Although BE effectively edits epitopes, complex bystander products are a concern. To enhance precision, we optimized prime editing, increasing the editing efficiency from 5.9% to 78.9% in HSPCs. Epitope-modified cells were resistant to CAR-T lysis while retaining normal differentiation and function. Furthermore, BE- or PE-edited HSPCs infused into humanized mice endowed myeloid lineages with selective resistance to CAR-T immunotherapy, demonstrating a proof-of-concept strategy for treating relapsed AML., Competing Interests: Declaration of interests Y.Z., G.-H.C., and R.-J.J. filed a PCT patent related to this work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chimeric antigen receptor T cell (CAR-T) therapy has emerged as a revolutionary approach in the treatment of malignancies. Despite its remarkable successes, this field continues to grapple with challenges such as scalability, safety concerns, limited therapeutic effect, in vivo persistence, and the need for precise control over CAR expression. In the post-pandemic era of COVID-19 vaccine immunization, the application of messenger RNA (mRNA) encapsulated within lipid nanoparticles (LNPs) has recently garnered significant attention as a potential solution to address these challenges. This review delves into the dynamic landscape of mRNA-LNP technology and its potential implications for CAR-engineered immune cell-based immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

Cytotoxic T lymphocytes (CTLs) play a crucial role in cancer rejection. However, CTLs encounter dysfunction and exhaustion in the immunosuppressive tumor microenvironment (TME). Although the reactive oxygen species (ROS)-rich TME attenuates CTL function, the underlying molecular mechanism remains poorly understood. The nuclear factor erythroid 2-related 2 (Nrf2) is the ROS-responsible factor implicated in increasing susceptibility to cancer progression. Therefore, we examined how Nrf2 is involved in anti-tumor responses of CD8 + T and chimeric antigen receptor (CAR) T cells in the ROS-rich TME. Here, we demonstrated that tumor growth in Nrf2 -/- mice was significantly controlled and was reversed by T cell depletion and further confirmed that Nrf2 deficiency in T cells promotes anti-tumor responses using an adoptive transfer model of antigen-specific CD8 + T cells. Nrf2-deficient CTLs are resistant to ROS, and their effector functions are sustained in the TME. Furthermore, Nrf2 knockdown in human CAR-T cells enhanced the survival and function of intratumoral CAR-T cells in a solid tumor xenograft model and effectively controlled tumor growth. ROS-sensing Nrf2 inhibits the anti-tumor T cell responses, indicating that Nrf2 may be a potential target for T cell immunotherapy strategies against solid tumors., Competing Interests: Declaration of interests C.H. received funding from NeoImmuneTech, Inc. D.C., S.-K.I., and B.H.L. are currently employed by NeoImmuneTech, Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Adoptive cell therapy using chimeric antigen receptor (CAR) T cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has been limited in solid tumors. One key factor for this is cancer-associated fibroblasts (CAFs) that modulate the tumor microenvironment (TME) to inhibit T cell infiltration and induce "T cell dysfunction." Additionally, the sparsity of tumor-specific antigens (TSA) and expression of CAR-directed tumor-associated antigens (TAA) on normal tissues often results in "on-target off-tumor" cytotoxicity, raising safety concerns. Using TALEN-mediated gene editing, we present here an innovative CAR T cell engineering strategy to overcome these challenges. Our allogeneic "Smart CAR T cells" are designed to express a constitutive CAR, targeting FAP + CAFs in solid tumors. Additionally, a second CAR targeting a TAA such as mesothelin is specifically integrated at a TCR signaling-inducible locus like PDCD1. FAPCAR-mediated CAF targeting induces expression of the mesothelin CAR, establishing an IF/THEN-gated circuit sensitive to dual antigen sensing. Using this approach, we observe enhanced anti-tumor cytotoxicity, while limiting "on-target off-tumor" toxicity. Our study thus demonstrates TALEN-mediated gene editing capabilities for design of allogeneic IF/THEN-gated dual CAR T cells that efficiently target immunotherapy-recalcitrant solid tumors while mitigating potential safety risks, encouraging clinical development of this strategy., Competing Interests: Declaration of interests S.Dharani, H.C., A.J., J.V., P.D., L.P., and S.Das are current employees and equity holders at Cellectis. TALEN is a Cellectis patented technology. J.P.F. is a former Cellectis employee and is a current employee of Bristol Myers Squibb., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: One of the significant challenges for cell therapies, such as chimeric antigen receptor (CAR)-T cell therapy, is the poor infiltration of immune cells into tumor tissues. CAR-monocytes/macrophages (CAR-M) are promising therapies because of their enrichment in the tumor microenvironment. Thus, we constructed a novel CAR-M to facilitate the infiltration of T cells and other immune cells., Methods: The suicide gene inducible caspase-9 ( iCasp9 ) and anti-erb-b2 receptor tyrosine kinase 2 (HER2) CAR elements were transfected into THP1 (an immortalized human monocyte cell line) by lentivirus. The suicide efficiency and specific anti-tumor efficacy were assessed using flow cytometry, inCucyte, and tumor-bearing BALB/c-nude mouse models. The activation of related signaling pathways in CAR-THP1 activation was explored by transcriptome sequencing. Finally, the synergistic therapeutic efficacy of CAR-THP1 combined with RAK cell treatment was demonstrated in tumor-bearing NOD.CB17-Prkdc scid Il2rg tm1 /Bcgen mouse models., Results: We developed a novel CAR-THP1, which incorporated iCasp9, CD3ζ, and CD147 intracellular segments, based on the first-generation HER2-CAR backbone. By constructing and comparing a series of CARs with different permutations, CAR-CD3ζ-CD147-iCasp9-THP1 was selected as the optimal combination. CAR-CD3ζ-CD147-iCasp9-THP1 initiated suicide quickly and efficiently under the control of iCasp9 gene, which enabled us to achieve controlled proliferation of CAR-THP1. CAR-THP1 also exhibited robust specific anti-tumor efficacy independently of T cells in vitro and in vivo . Through transcriptional sequencing, we found that CAR-THP1 tended to differentiate into the M1 phenotype and bridged innate and adaptive immunity. A combination of CAR-THP1 and Retronectin actived killer cells (RAKs) showed better therapeutic efficiency, as the metalloproteinases (MMPs) secreted by CAR-THP1 facilitated the degradation of the dense tumor matrix. This further assisted intratumoral infiltration of T cells and augmented the anti-tumor immune response., Conclusion: CAR-THP1 might be effective against HER2-positive tumor cells and has great potential for combination therapy with other immune cells., (Copyright © 2024 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

Surgical resection is a primary treatment option for patients with triple-negative breast cancer (TNBC), but it is associated with a high rate of postoperative local and metastatic relapse. Although chimeric antigen receptor-engineered NK (CAR-NK) cell therapy can specifically recognize and eradicate tumor cells, its therapeutic potency toward TNBCs is markedly suppressed by the hostile tumor microenvironment, which restricts the infiltration, survival, and effector functions of CAR-NK cells inside tumor masses. In this study, HER1-overexpressing TNBC-targeted CAR-NK (HER1-CAR-NK) cells were genetically engineered with catalase to endow them with tolerance toward the high levels of oxidative stress and hypoxia inside TNBC tumors through the catalytic decomposition of hydrogen peroxide, which is a principle reactive oxygen species inside tumors, into O2. We refer to these cells as HER1-CAR-CAT-NK cells. Upon intratumoral fixation with an injectable alginate hydrogel, HER1-CAR-CAT-NK cells enabled sustained tumor hypoxia attenuation and exhibited markedly enhanced persistence and effector functions inside TNBC tumors. As a result, locoregional HER1-CAR-CAT-NK cell therapy not only inhibited the growth of local primary residual tumors but also elicited systemic antitumor activity to suppress the growth of distant tumors. This study highlights that genetic engineering of HER1-CAR-NK cells with catalase is a promising strategy to suppress the postoperative local and distant relapse of TNBC tumors., (©2024 American Association for Cancer Research.)

Intraperitoneal cellular immunotherapy with CAR-redirected lymphocytes is an intriguing approach to target peritoneal carcinomatosis (PC) from ovarian cancer (OC), which is currently evaluated in clinical trials. PC displays a composite structure with floating tumor cells within ascites and solid-like masses invading the peritoneum. Therefore, a comprehensive experimental model is crucial to optimize CAR-cell therapies in such a peculiar environment. Here, we explored the activity of cytokine-induced killer lymphocytes (CIK), redirected by CAR against mesothelin (MSLN-CAR.CIK), within reductionistic 3D models resembling the structural complexity of both liquid and solid components of PC. MSLN-CAR.CIK effectively killed and were functionally efficient against OC targets. In a "floating-like" 3D context with floating OC spheroids, both tumor localization and killing by MSLN-CAR.CIK were significantly boosted by fluid flow. In a "solid-like" context, MSLN-CAR.CIK were recruited through the extracellular matrix on embedded tumor aggregates, with variable kinetics depending on the effector-target distance. Furthermore, MSLN-CAR.CIK penetrated the inner levels of OC spheroids exerting effective tumor killing. Our findings provide currently unknown therapeutically relevant information on intraperitoneal approaches with CAR.CIK, supporting further developments and improvements for clinical studies in the context of locoregional cell therapy approaches for patients with PC from OC., (© 2024. The Author(s).)

Patients with advanced gastric and colorectal cancers have limited treatment options. Since mesothelin is highly expressed in these tumour types, we evaluated the therapeutic benefits of anti-mesothelin hYP218 CAR T cells alone, and in combination with anti-PD1 antibody, pembrolizumab. GEPIA analysis was performed using human gastric (n = 408) and colon cancer tumours (n = 275) in TCGA database, to evaluate mRNA expression of mesothelin, compared to normal tissues. Mesothelin expression in gastric and colorectal cancer cell-lines (n = 5) was analysed using flow cytometry. In vitro efficacy by hYP218 CAR T cells was tested by cytotoxicity and cytokine release assays. In vivo anti-tumour efficacy of hYP218 CAR T cells alone, and in combination with pembrolizumab, was evaluated in NSG mice bearing human gastric (HGC27) and colorectal (SW48) tumour xenografts. Additionally, hYP218 CAR-T cell persistence, activation and exhaustion marker-expression were studied. Mesothelin expression was significantly higher in gastric and colon cancer biopsies compared to normal tissues (p < .005). Mesothelin expression in gastric and colon cancer cell lines ranged from 10 000 to 70 000 molecules per cell. hYP218 CAR T cells demonstrated strong cytotoxic activity at low effector to target ratio, ranging from 0.24 to 1.0. In NSG mouse-models, hYP218 CAR T cells demonstrated anti-tumour efficacy and persisted in the tumour microenvironment in a functional state at day 40 posttreatment with expression of activation markers CD39 and CD69, increased production of IFN-γ and TNF-α and ability to kill tumour cells in vitro when isolated from tumours. There was increased PD1 expression. In combination with pembrolizumab, hYP218 CAR T cells led to slower tumour growth in NSG mice bearing large but not small HGC27 tumours. Anti-tumour efficacy of hYP218 CAR T cells is due to increased accumulation of activated CAR T cells in the tumour and combination with pembrolizumab resulted in improvement in anti-tumour activity of large established tumours. HIGHLIGHTS: Mesothelin expression is significantly higher in gastric and colorectal cancers than normal tissues. hYP218 CAR T cells demonstrate strong anti-tumour activity against mesothelin-positive gastric and colorectal carcinomas. Activated hYP218 CAR T cells persist in the tumour microenvironment and retain their cytotoxic activity. Addition of pembrolizumab in larger tumours enhance CAR T cell efficacy., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs) Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells Support Protocol 1: Plasmid amplification Support Protocol 2: Lentivirus preparation Support Protocol 3: Lentivirus titration., (© 2024 Wiley Periodicals LLC.)

Introduction: Acute myeloid leukemia (AML) is a complex and heterogeneous disease characterized by an aggressive clinical course and limited efficacious treatment options in the relapsed/refractory (R/R) setting. Chimeric antigen receptor (CAR)-modified T (CAR-T) cell immunotherapy is an investigational treatment strategy for R/R AML that has shown some promise. However, obstacles to successful CAR-T cell immunotherapy for AML remain., Areas Covered: In analyses of clinical trials of CAR-T cell therapy for R/R AML, complete responses without measurable residual disease have been reported, but the durability of those responses remains unclear. Significant barriers to successful CAR-T cell therapy in AML include the scarcity of suitable tumor-target antigens (TTA), inherent T cell functional deficits, and the immunoinhibitory and hostile tumor microenvironment (TME). This review will focus on these barriers to successful CAR-T cell therapy in AML, and discuss scientific advancements and evolving strategies to overcome them., Expert Opinion: Achieving durable remissions in R/R AML will likely require a multifaceted approach that integrates advancements in TTA selection, enhancement of the intrinsic quality of CAR-T cells, and development of strategies to overcome inhibitory mechanisms in the AML TME.

Mounting effective immunity against pathogens and tumours relies on the successful metabolic programming of T cells by extracellular fatty acids 1-3 . Fatty-acid-binding protein 5 (FABP5) has a key role in this process by coordinating the efficient import and trafficking of lipids that fuel mitochondrial respiration to sustain the bioenergetic requirements of protective CD8 + T cells 4,5 . However, the mechanisms that govern this immunometabolic axis remain unexplored. Here we report that the cytoskeletal organizer transgelin 2 (TAGLN2) is necessary for optimal fatty acid uptake, mitochondrial respiration and anticancer function in CD8 + T cells. TAGLN2 interacts with FABP5 to facilitate its cell surface localization and function in activated CD8 + T cells. Analyses of ovarian cancer specimens revealed that endoplasmic reticulum (ER) stress responses induced by the tumour microenvironment repress TAGLN2 in infiltrating CD8 + T cells, thereby enforcing their dysfunctional state. Restoring TAGLN2 expression in ER-stressed CD8 + T cells increased their lipid uptake, mitochondrial respiration and cytotoxic capacity. Accordingly, chimeric antigen receptor T cells overexpressing TAGLN2 bypassed the detrimental effects of tumour-induced ER stress and demonstrated therapeutic efficacy in mice with metastatic ovarian cancer. Our study establishes the role of cytoskeletal TAGLN2 in T cell lipid metabolism and highlights the potential to enhance cellular immunotherapy in solid malignancies by preserving the TAGLN2-FABP5 axis., Competing Interests: Competing interests: J.R.C.-R. holds patents on the targeting of ER stress responses for the treatment of disease, as well as on the use of immune modulators for OC therapy. J.R.C.-R. is a scientific consultant for Moderna, Immagene, Autoimmunity Biologic Solutions, and Emerald Bioventures, and holds stock options in Vescor Therapeutics. D.Z. reports institutional grants from Merck, Genentech, AstraZeneca, Plexxikon, and Synthekine, and personal fees from AstraZeneca, Xencor, Memgen, Takeda, Synthekine, Immunos, Tessa Therapeutics, Miltenyi, and Calidi Biotherapeutics. D.Z. owns a patent on use of oncolytic Newcastle Disease Virus for cancer therapy. J.R.C.-G. has stock options in Compass Therapeutics, Anixa Biosciences, and Alloy Therapeutics; receives licensing fees from Anixa Biosciences for the patent of FSHCER T cells; receives honorarium from Alloy Therapeutics; has intellectual property with Compass Therapeutics and Anixa Biosciences; and is co-founder of Cellepus Therapeutics, a company that develops allogeneic γδ CAR T cells. All other authors declare no potential conflicts of interest., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Glycosaminoglycans are often deprioritized as targets for synthetic immunotherapy due to the complexity of glyco-epitopes and limited options for obtaining specific subtype binding. Solid tumors express proteoglycans that are modified with oncofetal chondroitin sulfate (CS), a modification normally restricted to the placenta. Here, we report the design and functionality of transient chimeric antigen receptor (CAR) T cells with selectivity to oncofetal CS. Following expression in T cells, the CAR could be "armed" with recombinant VAR2CSA lectins (rVAR2) to target tumor cells expressing oncofetal CS. While unarmed CAR T cells remained inactive in the presence of target cells, VAR2-armed CAR T cells displayed robust activation and the ability to eliminate diverse tumor cell types in vitro. Cytotoxicity of the CAR T cells was proportional to the concentration of rVAR2 available to the CAR, offering a potential molecular handle to finetune CAR T cell activity. In vivo, armed CAR T cells rapidly targeted bladder tumors and increased the survival of tumor-bearing mice. Thus, our work indicates that cancer-restricted glycosaminoglycans may be exploited as potential targets for CAR T cell therapy., Competing Interests: Disclosure and competing interests statement MD as the corresponding author certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (e.g., employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: MD, AS, and PHS are co-founders of, and shareholders in, VAR2 Pharmaceuticals. NAN and TG are consultants for VAR2 Pharmaceuticals. VAR2 Pharmaceuticals is a biotechnology company that specializes in the therapeutic development of the VAR2CSA technology (www.var2pharma.com). The remaining authors declare no competing interests., (© 2024. The Author(s).)

Background: Macrophages engineered with chimeric antigen receptors (CAR) are suitable for immunotherapy based on their immunomodulatory activity and ability to infiltrate solid tumours. However, the production and application of genetically edited, highly effective, and mass-produced CAR-modified macrophages (CAR-Ms) are challenging., Methods: Here, we used homology-independent targeted insertion (HITI) for site-directed CAR integration into the safe-harbour region of human pluripotent stem cells (hPSCs). This approach, together with a simple differentiation protocol, produced stable and highly effective CAR-Ms without heterogeneity., Findings: These engineered cells phagocytosed cancer cells, leading to significant inhibition of cancer-cell proliferation in vitro and in vivo. Furthermore, the engineered CARs, which incorporated a combination of CD3ζ and Megf10 (referred to as FRP5 Mζ ), markedly enhanced the antitumour effect of CAR-Ms by promoting M1, but not M2, polarisation. FRP5 Mζ promoted M1 polarisation via nuclear factor kappa B (NF-κB), ERK, and STAT1 signalling, and concurrently inhibited STAT3 signalling even under M2 conditions. These features of CAR-Ms modulated the tumour microenvironment by activating inflammatory signalling, inducing M1 polarisation of bystander non-CAR macrophages, and enhancing the infiltration of T cells in cancer spheroids., Interpretation: Our findings suggest that CAR-Ms have promise as immunotherapeutics. In conclusion, the guided insertion of CAR containing CD3ζ and Megf10 domains is an effective strategy for the immunotherapy of solid tumours., Funding: This work was supported by KRIBB Research Initiative Program Grant (KGM4562431, KGM5282423) and a Korean Fund for Regenerative Medicine (KFRM) grant funded by the Korean government (Ministry of Science and ICT,Ministry of Health and Welfare) (22A0304L1-01)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The potential of macrophage-mediated phagocytosis as a cancer treatment is promising. Blocking the CD47-SIRPα interaction with a CD47-specific antibody significantly enhances macrophage phagocytosis. However, concerns regarding their toxicity to nontumor cells remain substantial. Here, we engineered chimeric antigen receptor macrophages (CAR-Ms) by fusing a humanized single-chain variable fragment with FcγRIIa and integrating short hairpin RNA to silence SIRPα, thereby disrupting the CD47-SIRPα signaling pathway. These modified CAR-shSIRPα-M cells exhibited an M1-like phenotype, superior phagocytic function, substantial cytotoxic effects on HER2-positive tumor cells, and the ability to eliminate patient-derived organoids. In vivo, CAR-M cells significantly inhibited tumor growth and prolonged survival in tumor-bearing mice. Notably, CAR-shSIRPα-M cells enhanced cytotoxic T-cell infiltration into tumors, thereby enhancing the antitumor response in both the humanized immune system mouse model and immunocompetent mice. Mechanistically, SIRPα inhibition activated inflammatory pathways and the cGAS-STING signaling cascade in CAR-M cells, leading to increased production of proinflammatory cytokines, reactive oxygen species, and nitric oxide, thereby enhancing their antitumor effects. These findings underscore the potential of SIRPα inhibition as a novel strategy to increase the antitumor efficacy of CAR-M cells in cancer immunotherapy, particularly against solid tumors., (© 2024. The Author(s).)

Chimeric antigen receptor (CAR) T-cell therapy has achieved significant success in achieving durable and potentially curative responses in patients with hematological malignancies. CARs are tailored fusion proteins that direct T cells to a specific antigen on tumor cells thereby eliciting a targeted immune response. The approval of several CD19-targeted CAR T-cell therapies has resulted in a notable surge in clinical trials involving CAR T cell therapies for hematological malignancies. Despite advancements in understanding response mechanisms, resistance patterns, and adverse events associated with CAR T-cell therapy, the translation of these insights into robust clinical efficacy has shown modest outcomes in both clinical trials and real-world scenarios. Therefore, the assessment of CAR T-cell functionality through rigorous preclinical studies plays a pivotal role in refining therapeutic strategies for clinical applications. This review provides an overview of the various in vitro and animal models used to assess the functionality of CAR T-cells. We discuss the findings from preclinical research involving approved CAR T-cell products, along with the implications derived from recent preclinical studies aiming to optimize the functionality of CAR T-cells. The review underscores the importance of robust preclinical evaluations and the need for models that accurately replicate human disease to bridge the gap between preclinical success and clinical efficacy., Competing Interests: Declaration of competing interest Dr. Melenhorst has patents related to Biomarkers and manufacturing of chimeric antigen receptor-engineered T cells. The remaining authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Natural killer (NK) cells play a vital role in innate immunity and show great promise in cancer immunotherapy. Traditional sources of NK cells, such as the peripheral blood, are limited by availability and donor variability. In addition, in vitro expansion can lead to functional exhaustion and gene editing challenges. This study aimed to harness induced pluripotent stem cell (iPSC) technology to provide a consistent and scalable source of NK cells, overcoming the limitations of traditional sources and enhancing the potential for cancer immunotherapy applications. We developed human placental-derived iPSC lines using reprogramming techniques. Subsequently, an optimized two-step differentiation protocol was introduced to generate high-purity NK cells. Initially, iPSCs were differentiated into hematopoietic-like stem cells using spin-free embryoid bodies (EBs). Subsequently, the EBs were transferred to ultra-low attachment plates to induce NK cell differentiation. iPSC-derived NK (iNK) cells expressed common NK cell markers (NKp46, NKp30, NKp44, CD16 and eomesodermin) at both RNA and protein levels. iNK cells demonstrated significant resilience to cryopreservation and exhibited enhanced cytotoxicity. The incorporation of a chimeric antigen receptor (CAR) construct further augmented their cytotoxic potential. This study exemplifies the feasibility of generating iNK cells with high purity and enhanced functional capabilities, their improved resilience to cryopreservation and the potential to have augmented cytotoxicity through CAR expression. Our findings offer a promising pathway for the development of potential cellular immunotherapies, highlighting the critical role of iPSC technology in overcoming challenges associated with traditional NK cell sources., (© 2024 The Author(s). Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Background Aims: Chimeric antigen receptor T (CAR-T) cells targeting single antigens show limited activity against solid tumors due to poor T cell persistence, low efficiency infiltration, and exhaustion together with heterogeneous tumor-associated antigen (TAA) expression. This is also true in high-risk neuroblastoma (HRNB), a lethal pediatric extracranial malignancy. To overcome these obstacles, a combinational strategy using GD2-specific and GPC2-specific CAR-T cells was developed to improve immunotherapeutic efficacy., Methods: We individually developed GD2-specific and GPC2-specific CARs containing a selective domain (sCAR) which was a peptide of 10 amino acids derived from human nuclear autoantigen La/SS-B. These constructs allowed us to generate two different HRNB antigen-specific CAR-T cells with enhanced biological activity through stimulating sCAR-engrafted T cells via a selective domain-specific monoclonal antibody (SmAb). Binding affinity and stimulation of GD2- and GPC2-specific sCARs by SmAb were measured, and transient and persistent anti-tumor cytotoxicity of GD2sCAR-T and GPC2sCAR-T cells were quantified in neuroblastoma cell lines expressing different TAA levels. The anti-tumor pharmaceutical effects and cellular mechanisms mediated by single or combinational sCAR-T cells were evaluated in vitro and in vivo., Results: GD2- and GPC2-specific sCARs had antigen-specific binding affinity similar to their parental counterparts and were recognized by SmAb. SmAb-mediated stimulation selectively activated sCAR-T proliferation and increased central memory T cells in the final products. SmAb-stimulated sCAR-T cells had enhanced transient cytolytic activity, and combination therapy extended long-term anti-tumor activity in vitro through TNF-α and IL-15 release. Stimulated sCAR-T cells overcame heterogeneous antigen expression in HRNB, and the multi-TAA-targeting strategy was especially efficacious in vivo, inducing apoptosis through the caspase-3/PARP pathway and inhibiting the release of several tumor-promoting cytokines., Conclusions: These data suggest that combined targeting of multiple TAAs is a promising strategy to overcome heterogenous antigen expression in solid tumors and extend CAR-T cell persistence for HRNB immunotherapy., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The success of chimeric antigen receptor (CAR) T cell therapy in treating several hematopoietic malignancies has been difficult to replicate in solid tumors, in part because of T cell exhaustion and eventually dysfunction. To counter T cell dysfunction in the tumor microenvironment, we metabolically armored CAR T cells by engineering them to secrete interleukin-10 (IL-10). We show that IL-10 CAR T cells preserve intact mitochondrial structure and function in the tumor microenvironment and increase oxidative phosphorylation in a mitochondrial pyruvate carrier-dependent manner. IL-10 secretion promoted proliferation and effector function of CAR T cells, leading to complete regression of established solid tumors and metastatic cancers across several cancer types in syngeneic and xenograft mouse models, including colon cancer, breast cancer, melanoma and pancreatic cancer. IL-10 CAR T cells also induced stem cell-like memory responses in lymphoid organs that imparted durable protection against tumor rechallenge. Our results establish a generalizable approach to counter CAR T cell dysfunction through metabolic armoring, leading to solid tumor eradication and long-lasting immune protection., Competing Interests: Competing interests Y.G., L.T. and Y.Z. are inventors on patents related to the technology described in this paper. L.T. and Y.G. are cofounders, share-holders and advisors for Leman Biotech. The interests of L.T. were reviewed and managed by EPFL. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)